Cystic fibrosis (CF) is a disease that arises due to mutations in the gene that codes for cystic fibrosis transmembrane conductance regulator (CFTR), which is a membrane protein involved in chloride ion secretion [1]. Although most cystic fibrosis patients develop chronic progressive disease of the respiratory system, the disease can cause damage to many other organs and tissues. For instance, pancreatic dysfunction, hepatobiliary and genitourinary diseases are all common manifestations of the cystic fibrosis disorder. The diverse array of symptoms and disorders caused by cystic fibrosis have made treatment of the disorder a difficult task. Many treatment modes have focused on improving the clinical symptoms of the particular organ affected in the patient, such as antibiotic treatments, improved nutritional care, and physiotherapy. Additionally, therapies have been developed which attempt to counteract the biochemical basis of the genetic disease, such as gene therapy with CFTR genes. None of these treatment methods, however, has been entirely successful in the treatment of cystic fibrosis.
The most serious consequence of cystic fibrosis (CF) is Pseudomonas aeruginosa lung infection, which by itself accounts for almost 90% of the morbidity and mortality in CF [3]. By age 12, 60-90% of CF patients are infected with P. aeruginosa, and most die before age 30 [3]. Pathogens such as S. aureus and nontypable H. influenza are also commonly isolated from the respiratory tract of CF patients, but only P. aeruginosa infection has been associated with the progressive decline in pulmonary function in these patients [4-6].
Progressive loss of pulmonary function over many years due to chronic infection with mucoid P. aeruginosa is the hallmark of CF, and yet the connection between lung infection and defects in chloride ion conductance have remained elusive. Smith et al. [2] recently reported defective bacterial killing by fluid obtained from airway epithelial cell cultures of CF patients. Smith et al. reported that this phenomenon was due to the inhibition of an unidentified antimicrobial factor resulting from increased levels of sodium chloride in the airway epithelial fluid.
Many of the severe cases of CF are associated with CFTR mutations leading to greatly reduced to no cell-surface expression of CFTR. The most prevalent of the CFTR mutations is the deletion of phenylalanine 508. Mutant CFTR genes having a deleted phenylalanine 508 are referred to as xcex94F508. xcex94F508 accounts for approximately 70% of the cystic fibrosis alleles. The xcex94F508 mutation has been associated with elevated sweat chloride levels and severe physiological effects such as chronic pulmonary disease in many patients.
Pier et al. has proposed that ingestion and clearance of P. aeruginosa by epithelial cells could be one mechanism by which the epithelial cells protect the lungs against infection [7]. The study reported that ingestion and clearance of P. aeruginosa was compromised in a cell line derived from a patient with the xcex94F508 CFTR mutation and was specific for P. aeruginosa among the respiratory pathogens evaluated [7]. Expression of wild-type CFTR by transection, or induction of membrane expression of mutant xcex94F508 CFTR by growth of cells at 26xc2x0 C., increased P. aeruginosa ingestion. Inhibition of ingestion of P. aeruginosa by cells in neonatal mouse lungs increased the total bacterial load in the lungs [7]. These studies showed that CFTR modulated this epithelial cell process but did not specifically indicate how CFTR was involved in the process.
The invention involves the discovery that P. aeruginosa binds to the cystic fibrosis transmembrane conductance regulator (CFTR) (SEQ.ID.NO.1) and, in particular, that the core portion of the lipopolysaccharide of P. aeruginosa binds the CFTR. The invention also involves the discovery that contacting cells expressing the CFTR with the core portion of the lipopolysaccharide of P. aeruginosa results in upregulation of the CFTR. Upregulation of the CFTR in epithelial mucosa further was discovered to result in better clearance of P. aeruginosa, and, therefore, methods for preventing, inhibiting or eradicating Pseudomal infection are provided, including subjects having cystic fibrosis. In general, these discoveries have led to methods and products using fragments of the lipopolysaccharide of P. aeruginosa and using fragments of the CFTR in the manufacture of pharmaceutical products, diagnostic products, research tools, and methods relating hereto.
According to one aspect of the invention, a method for upregulating CFTR expression in the tissue of a subject is provided. A CFTR expression regulator is administered to a subject in need of upregulation of CFTR expression, in an amount effective to increase CFTR expression in the tissue of the subject. The CFTR expression regulator is an isolated polysaccharide that is an LPS core moiety comprising 
wherein X is selected from the group consisting of glucose, glucose-rhamnose and H; Y is selected from the group consisting of rhammose and H; and Z is selected from the group consisting of glucose and H;
A preferred polysaccharide is an LPS core moiety comprising 
One particularly useful polysaccharide according to the invention comprises 
Another particularly useful polysaccharide according to the invention is: 
The foregoing preferred molecules can be isolated CFTR receptor-binding fragments of lipopolysaccharides of P. aeruginosa. 
In one embodiment of the invention, the subject has a condition predisposing the subject to Pseudomal infection. In another embodiment of the invention, the subject has a Pseudomal infection. In one important embodiment of the invention, the subject has a defective cystic fibrosis transmembrane conductance regulator gene.
According to another aspect of the invention, a pharmaceutical preparation is provided. The pharmaceutical preparation is a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a CFTR expression regulator. The CFTR expression regulator is as described above. The pharmaceutical preparation can be sterile and can be formulated in a unit dosage in an amount effective for treating Pseudomal infection. As used herein, xe2x80x9ctreatingxe2x80x9d means preventing the onset of, slowing the progression of, or eradicating the existence of the condition being treated, such as a Pseudomal infection. The pharmaceutical preparation can be formulated as any suitable preparation, including a preparation suitable for inhalation or a preparation suitable for injection.
According to another aspect of the invention, compositions of matter are provided. The compositions are covalent conjugates. One composition is a covalent conjugate of a lipid biocompatible with a human subject and a polysaccharide. The polysaccharide is as described above. In one embodiment, the lipid portion of the conjugate is inserted within the wall of a liposome and the polysaccharide is exposed on the surface of the liposome. The liposome contains a bioactive agent.
Another composition is a covalent conjugate of a bioactive agent and a polysaccharide. Again, the polysaccharide is as described above.
The foregoing covalent conjugates are useful in delivering bioactive agents to cells and/or tissues expressing a CFTR. Thus, methods are provided for delivering a bioactive agent to a tissue expressing a cystic fibrosis transmembrane conductance regulator to treat a condition susceptible to treatment by the bioactive agent. A bioactive agent coupled to a polysaccharide is administered to a subject in need of such treatment, in an amount effective for treating the condition. The polysaccharide is as described above. The bioactive agent can be noncovalently or covalently linked to the polysaccharide, or the bioactive agent can be contained in a liposome comprising a lipid biocompatible with a human subject, wherein the polysaccharide is covalently coupled to the lipid.
As a result of the discovery that the CFTR binds the lipopolysaccharide of P. aeruginosa, methods and products involving the use of CFTR fragments are provided.
According to one aspect of the invention, a composition of matter is provided. The composition is a covalent conjugate of an anti-Pseudomonas drug and CFTR or a lipopolysaccharide-binding fragment of a cystic fibrosis transmembrane conductance regulator. The lipopolysaccharide-binding fragment of a CFTR preferably comprises at least four consecutive amino acids of Sequence ID No. 3, and can comprise at least five, six, seven or eight consecutive amino acids of Sequence ID. No. 3.
According to another aspect of the invention, an isolated polypeptide is provided. The isolated polypeptide is a lipopolysaccharide-binding fragment of a cystic. fibrosis transmembrane conductance regulator. The fragment preferably comprises at least four consecutive amino acids of Sequence ID. No. 3, and comprise at least five, six, seven or eight consecutive amino acids of Sequence ID. No. 3. Even more preferably, the fragment is between seven and twelve amino acids in length.
According to another aspect of the invention, methods for targeting an anti-Pseudomonas drug to a Pseudomonas microorganism is provided. The method involves contacting the environment of the Pseudomonas with a lipopolysaccharide-binding fragment as described above coupled to an anti-pseudomonas drug.
According to still another aspect of the invention, isolated nucleic acids are provided. The isolated nucleic acids encode the lipopolysaccharide-binding fragments of CFTR described above.
These and other aspects of the invention are described in greater detail below. It is noted that the isolated polysaccharides of the invention, in preferred embodiments, have the more detailed structures as described below in connection with the detailed description of the invention. The se particular structures are considered important aspects of the invention.
The invention involves the discovery that P. aeruginosa binds to CFTR and, more particularly, that the lipopolysaccharide (LPS) core of P. aeruginosa binds to CFTR Surprisingly, it was discovered that contacting cells expressing the CFTR with the P. aeruginosa LPS core resulted in upregulation of the expression of CFTR. It also was discovered, surprisingly, that such upregulation results in improved uptake of P. aeruginosa by such cells and improved clearance of P. aeruginosa by such cells, thereby providing the basis of a therapy using the core of P. aeruginosa LPS to inhibit, prevent or otherwise treat infection by P. aeruginosa. Mammals pretreated with LPS core fragments are less susceptible to infection by P. aeruginosa. They also recover more quickly from infection by P. aeruginosa than animals without such pretreatment. As a result of the foregoing discoveries, methods and products are provided that make use of CFTR binding fragments of P. aeruginosa LPS cores. Methods and products also are provided that make use of LPS binding-fragments of cystic fibrosis transmembrane conductance regulators. (The mRNA and amino acid sequences of CFTR are provided in SEQ.ID.NO.1 and SEQ.ID.NO.2, respectively.)
The methods and products of the invention are useful in connection with cells, microorganisms and subjects.
As used herein, a subject is a human, nonhuman primate, horse, cow, sheep, goat, dog, cat, or rodent.
As used herein in connection with polysaccharides and polypeptides, xe2x80x9cisolatedxe2x80x9d means essentially free of other substances with which the polysaccharides or polypeptides may be found in nature or in in vivo systems to an extent practical and appropriate for their intended use. The material is sufficiently pure and sufficiently flee of other biological materials so that it may be used in, for example, a pharmaceutical preparation. The material may be isolated using conventional techniques known to those of ordinary skill in the art. The material also may be prepared by synthetic chemistry using procedures known to those of ordinary skill in the art. Because an isolated material may be admixed with a pharmaceutically acceptable carrier(s) in a pharmaceutical preparation, the material may comprise only a small percentage by weight of the preparation; it nevertheless still is isolated as is meant herein. An isolated fragment of a polypeptide or polysaccharide also is a portion of the polypeptide or polysaccharide as found in nature, isolated from the remaining portion as found in nature.
According to one aspect of the invention, a method for upregulating CFTR expression in a tissue of a subject is provided. The method involves administering to a subject in need of such upregulation a CFTR expression regulator in an amount effective to increase CFTR expression in the tissue. The CFTR expression regulator is an isolated polysaccharide that is an LPS core moiety comprising: 
wherein X is selected from the group consisting of glucose, glucose-rhamnose and H; Y is selected from the group consisting of rhamnose and H; and Z is selected from the group consisting of glucose and H;
The entire core of the LPS of P. aeruginosa may be used, which consists essentially of the polysaccharide portion free of the lipid tail (which is somewhat toxic). For example, the isolated polysaccharide may be isolated from the O6 strain of P. aeruginosa, obtainable from the American Type Culture Collection, Rockville, Md. (ATCC) under excession no. 33354. Mutant strains of P. aeruginosa also are available, which strains contain the essential portions of the polysaccharide of the invention as described above, such as, for example, Pseudomonas strain O3, ATCC excession no. 33350.
The structure of O6 is believed to be as follows: 
The structure of O3 is believed to be as follows: 
Variants derived from other mutant strains or prepared by chemical synthesis are useful according to the invention. The following variants are specific examples: 
It is believed that the most preferable bonding configuration is 
Those of ordinary skill in the art will be able to identify other variants and modifications useful according to the invention Synthetic chemistry for constructing small polysaccharides is available. In addition, core polysaccharides can be derived from the many and various mutant strains of P. aeruginosa. These materials then may be simply tested for binding to the CFTR, the sequence of which is provided herein as Sequence ID. No. 1. The CFTR gene is the subject of gene therapy clinical protocols and has been studied extensively in various expression systems, many of which would be suitable for screening LPS core variant binding. The CFTR or the polysaccharide also could be bound to a substrate, such as a polystyrene plate. Screening experiments could involve direct measurement of the binding of the variant to CFTR if the variant were labeled, such as with a radioactive label or a florescent label. Likewise, the CFTR could be labeled in direct binding studies. Screening also can be carried out by measuring indirect binding such as in a competitive binding assay. Such assays could involve competition with P. aeruginosa binding to CFTR or with isolated core LPS of P. aeruginosa binding to CFTR. Those of ordinary skill in the art will readily know the details of such screening assays.
As mentioned above, the invention involves the surprising discovery that the polysaccharides of the invention upregulate CFTR expression and can result in increased uptake of P. aeruginosa and clearance of P. aeruginosa. Increased CFTR expression can be evaluated, for example, by measuring CFTR mRNA, by using antibodies against the CFTR, or by measuring LPS core binding to the CFTR Such measurements are well within the ability of those of ordinary skill in the art.
The invention is useful in treating a variety of conditions involving the CFTR. Most notably, the invention is useful whenever it is desirable to prevent, inhibit or halt infection by P. aeruginosa. This includes treating subjects having conditions that predispose the subjects to infection by P. aeruginosa, such as patients requiring medical intensive care (including surgical intensive care), patients with a basic compromise to the respiratory tract (such as by intubation), patients are immuno-compromised such as by anticancer chemotherapy treatment and patients with chronic obstructive pulmonary disease. Treatment can be prophylactic or can be concurrent with active infection. It is believed that frequent (daily) treatment will be most successful.
In the foregoing instances, the patients can have normal CFTRs. The invention is also useful when the patient""s CFTRs are not normal, such as with cystic fibrosis patients who have a defective CFTR gene. In some instances, it is believed that a defective CFTR still will bind P. aeruginosa, although weakly and inadequately. Thus, upregulation of a defective CFTR also will enhance clearance of P. aeruginosa, and, therefore, the polysaccharides of the invention are useful in treating subjects with defective CFTR genes.
It is noted that CFTR is expressed in a variety of tissues and is believed to have functions in addition to simply binding and clearing P. aeruginosa. The receptor is expressed in the gastrointestinal tract (intestinal epithelial cells), in the goblets within the respiratory tract, in genital tissue, in kidney tissue, in pancreas tissue, in liver tissue, and in corneal epithelial cells. Some of the negative effects of a defective CFTR gene are managed by diet, and it is believed that upregulation of the defective receptor would be of benefit in these situations. Negative effects of a defective CFTR include malabsorption in the gastrointestinal tract (managed by diet and by patients taking pancreatic supplements, but patients are still usually in the lower 5th percentile for weight and height); sterility in males (genitouriny tract); on occasion, diabetes (pancreas) and arthritis. The polysaccharides of the invention, therefore, are useful whenever it is desirable to upregulate CFTR expression, whether the CFTR gene is normal or defective.
The presence of the CFTR in a variety of tissues also has led to another aspect of the invention, that is the use of the isolated polysaccharides of the invention to target a bioactive molecule to a tissue expressing a CFTR. The CFTR binding fragment of a LPS of P. aeruginosa is used as a targeting moiety in an otherwise conventional manner, to target bioactive agents to tissues expressing the CFTR. In general the targeting moiety is coupled to the bioactive agent. The molecules may be directly coupled to one another, such as by conjugation or may be indirectly coupled to one another where, for example, the targeting moiety is on the surface of the liposome and the bioactive agent is contained within the liposome. Thus, the invention contemplates conjugates of the isolated polysaccharides of the invention with bioactive agents. If the molecules are linked to one another, then the targeting moiety is covalently or noncovalently bound to the bioactive agent in a manner that preserves the targeting specificity of the targeting moiety. As used herein, xe2x80x9clinkedxe2x80x9d or xe2x80x9clinkagexe2x80x9d means two entities are bound to one another by any physiochemical means. It is important that the linkage be of such a nature that it does not impair substantially the effectiveness of the bioactive molecule or the binding specificity of the targeting moiety. Keeping these parameters in mind, any linkage known to those of ordinary skill in the art may be employed, covalent or noncovalent. Such means and methods of linkage are well known to those of ordinary skill in the art.
Linkage according to the invention need not be direct linkage. The components of the compositions of the invention may be provided with functionalized groups to facilitate their linkage and/or linker groups may be interposed between the components of these compositions to facilitate their linkage. In addition, the components of the present invention may be synthesized in a single process, whereby the components could be regarded as one in the same entity. For example, a targeting moiety specific for a CFTR receptor could be synthesized together with the bioactive agent. These and other modifications are intended to be embraced by the present invention.
Specific examples of covalent bonds include those wherein bifunctional cross-linker molecules are used. The cross-inker molecules may be homobifunctional or heterobifunctional, depending upon the nature of the molecules to be conjugated. Homobifunctional cross-linkers have two identical reactive groups. Heterobifunctional cross-linkers have two different reactive groups that allow sequential conjugation reaction. Various types of commercially available cross-linkers are reactive with one or more of the following groups: primary amines, secondary amines, sulfhydriles, carboxyls, carbonyls and carbohydrates.
Non-covalent methods of conjugation also may be used to join the targeting moiety and the bioactive agent. Non-covalent conjugation may be accomplished by direct or indirect means including hydrophobic interaction, ionic interaction, intercalation, binding to major or minor grooves of a nucleic acid and other affinity interactions.
Covalent linkages may be noncleavable in physiological environments or cleavable in physiological environments, such as linkers containing disulfide bonds. Such molecules may resist degradation and/or may be subject to different intracellular transport mechanisms. One of ordinary skill in the art will be able to ascertain without undue experimentation the preferred bond for linking the targeting moiety and the bioactive agent, based on the chemical properties of the molecules being linked and the preferred characteristics of the bond.
For indirect linkage, the targeting moiety may be part of a particle, such as a liposome, which targets the liposome to tissues expressing the CFTR. The liposome, in turn, may contain the bioactive agent. The manufacture of liposomes containing bioactive agents is fully described in the literature. Many are based upon cholesteric molecules as starting ingredients and/or it phospholipids. They may be synthetically derived or isolated from natural membrane components. According to an embodiment of the invention, the polysaccharide which is the All targeting moiety is attached to a non-toxic, lipophilic anchor that is biocompatible with a human subject. Virtually any hydrophobic substance can be used, including cholesteric molecules, phospholipids and fatty acids preferably of medium chain length (12C-20C). Preferred are naturally occurring fatty acids of between 14 and 18 carbons in length. These molecules can be attached to the polysaccharides of the invention, with the lipophilic anchor inserting into the membrane of a liposome and the polysaccharide tethered on the surface of the liposome for targeting the liposome to the cell expressing the CFTR.
It is noted that lipid A normally is attached to native lipopolysaccharide of P. aeruginosa. Lipid A, however, is somewhat toxic, and it is preferred that the polysaccharides of the invention, if attached to lipophilic molecules, be attached to molecules which have a better toxicity profile than Lipid A. Useful lipophilic moieties may include any short to medium-chain length saturated fatty acid of the general composition CH3(CH2)nCOOH where n can range from 0-50. Preferably n will range from 1-20. In addition, the CH2 groups may be modified to be linked to each other through double-carbon bonds, making the fatty acid of the unsaturated category. Further modifications could include additional substituents such as hydroxyl (OH) groups added on to the fatty acid chain backbone to produce hydroxylated fatty acids. The substituents may be added to either the same side of the fatty acid chain (the trans-configuration) or to opposite sides of the fatty acid chain (the cis configuration). The lipophilic moieties may be attached to the targeting moieties as described above.
The bioactive agent may be virtually any bioactive agent useful in a cell expressing a CFTR Bioactive agents, as used herein, include diagnostic agents such as radioactive labels and fluorescent labels. Bioactive agents also include molecules affecting the metabolism of a cell expressing a CFTR, including peptides, nucleic acids, and other natural and synthetic drug molecules. Included are: adrenergic agent; adrenocortical steroid; adrenocortical suppressant; alcohol deterrent; aldosterone antagonist; amino acid; ammonia detoxicant; anabolic; analeptic; analgesic; androgen; anesthesia, adjunct to; anesthetic; anorectic; antagonist; anterior pituitary suppressant; anthelmintic; anti-acne agent; anti-adrenergic; anti-allergic; anti-amebic; anti-androgen; anti-anemic; anti-anginal; anti-anxiety; anti-arthritic; anti-asthmatic; anti-atherosclerotic; antibacterial; anticholelithic; anticholelithogenic; anticholinergic; anticoagulant; anticoccidal; anticonvulsant; antidepressant; antidiabetic; antidiarrheal; antidiuretic; antidote; anti-emetic; anti-epileptic; anti-estrogen; antifibrinolytic; antifungal; antiglaucoma agent; antihemophilic; antihemorrhagic; antihistamine; antihyperlipidemia; antihyperlipoproteinemic; antihypertensive; antihypotensive; anti-infective; anti-infective, topical; anti-inflammatory; antikeratinizing agent; antimalarial; antimicrobial; antimigraine; antimitotic; antimycotic, antinauseant, antineoplastic, antineutropenic, antiobessional agent; antiparasitic; antiparkinsonian; antiperistaltic, antipneumocystic; antiproliferative; antiprostatic hypertrophy; antiprotozoal; antipruritic; antipsychotic; antirheumatic; antischistosomal; antiseborrheic; antisecretory; antispasmodic; antithrombotic; antitussive; anti-ulcerative; anti-urolithic; antiviral; appetite suppressant; benign prostatic hyperplasia therapy agent; blood glucose regulator, bone resorption inhibitor; bronchodilator, carbonic anhydrase inhibitor; cardiac depressant; cardioprotectant; cardiotonic; cardiovascular agent; choleretic; cholinergic; cholinergic agonist; cholinesterase deactivator, coccidiostat; cognition adjuvant; cognition enhancer, depressant; diagnostic aid; diuretic; dopaminergic agent; ectoparasiticide; emetic; enzyme inhibitor; estrogen; fibrinolytic; fluorescent agent; free oxygen radical scavenger; gastrointestinal motility effector; glucocorticoid; gonad-stimulating principle; hair growth stimulant; hemostatic; histamine H2 receptor antagonists; hormone; hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive; imaging agent; immunizing agent; immunomodulator; inmmunoregulator, immunostimulant; immunosuppressant; impotence therapy adjuvant; inhibitor; keratolytic; LHRH agonist; liver disorder treatment; luteolysin; memory adjuvant; mental performance enhancer; mood regulator, mucolytic; mucosal protective agent; mydriatic; nasal decongestant; neuromuscular blocking agent; neuroprotective; NMDA, antagonist; non-hormonal sterol derivative; oxytocic; plasminogen activator, platelet activating factor antagonist; platelet aggregation inhibitor; post-stroke and post-head trauma treatment; potentiator; progestin; prostaglandin; prostate growth inhibitor; prothyrotropin; psychotropic; pulmonary surface; radioactive agent; regulator; relaxant; repartitioning agent; scabicide; sclerosing agent; sedative; sedative-hypnotic; selective adenosine A1 antagonist; serotonin antagonist; serotonin inhibitor, serotonin receptor antagonist; steroid; stimulant; suppressant; symptomatic multiple sclerosis; synergist; thyroid hormone; thyroid inhibitor; thyromimetic; tranquilizer; amyotrophic lateral sclerosis agent; cerebral ischemia agent; Paget""s disease agent; unstable angina agent; uricosuric; vasoconstrictor, vasodilator, vulnerary; wound healing agent; xanthine oxidase inhibitor.
In one preferred embodiment, a gene under the control of a promoter, preferably in a plasmid, is coupled to the targeting moiety for delivering the gene to the cell expressing the CFTR. In one particularly important embodiment, the gene is a normal CFTR gene and the methods and products of the invention are used to treat subjects with defective CFTR genes by gene therapy. A gene therapy contruct for CF, for example, may include either the cDNA sequence of CFTR incorporated into an appropriate expression system, or the genomic DNA sequence of CFTR including the coding exons and noncoding introns incorporated into an appropriate expression vector. It could also be a contruct containing only a portion of the CFTR that is needed to restore normal cellular function. For example, the first 150 amino acids are not needed for chloride ion conductance of the cell and this portion of CFTR could be produced from an appropriate cDNA or genomic DNA. Alternately, the portion of CFTR encoding the P. aeruginosa binding site (amino acids 103-118) could be expressed only in lung cells to promote resistance to infection since the rest of the molecule, which has ion-secretion properties, is not needed for resistance to infection. Antisense molecules can be delivered according to the methods of the invention as well. Thus, an important aspect of the invention is the targeting and delivery of oligonucleotides to cells expressing the CFTR.
Because of the discovery that CFTR binds P. aeruginosa LPS core, this has led to the further aspects of the invention related to the use of CFTR or CFTR fragments for therapeutic, diagnostic and research purposes as well as in vivo and in vitro methods relating thereto. Thus, according to another aspect of the invention, compositions of matters are provided that involve CFTR and fragments of the CFTR. In one aspect of the invention, a covalent conjugate of an anti-Pseudomonas drug and CFTR or a Pseudomonas lipopolysaccharide-binding fragment of CFTR is provided. The lipopolysaccharide-binding fragment comprises at least four consecutive amino acids of SEQ ID NO. 3. The fragment can comprise at least five consecutive amino acids, at least six consecutive amino acids, at least seven consecutive amino acids, or at least eight consecutive amino acids of SEQ ID NO. 3. SEQ ID NO. 3 consists of amino acids numbered 103-117 of the coding region of the CFTR. Thus, where at least four consecutive amino acids from SEQ ID NO. 3 are involved, there are twelve possibilities as follows: amino acids numbered 103-106, 104-107, 105-108, 106-109, 107-110, 108-111, 109-112, 110-113, 111-114, 112-115, 113-116 and 114-117. Where at least five consecutive amino acids are involved, there are eleven possibilities as follows: amino acids numbered 103-107, 104-108, 105-109, 106-110, 107-111, 108-112, 109-113, 110-114, 111-115, 112-116and 113-117. Where there are at least six consecutive amino acids involved, there are ten possibilities as follows: amino acids numbered 103-108, 104-109, 105-110, 106-111, 107-112, 108-113, 109-114, 110-115, 111-116 and 112-117. Where there are at least seven consecutive amino acids involved, there are nine possibilities as follows: amino acids numbered 103-109, 104-110, 105-111, 106-112, 107-113, 108-114, 109-115, 110-116and 111-117. Where there are eight consecutive amino acids involved, there are eight possibilities as follows: amino acids numbered 103-110, 104-111, 105-112, 106-113, 107-114, 108-115, 109-116 and 110-117.
It is believed that the optimal fragments will be between six and twelve amino acids in length, preferably between six and eight amino acids in length.
Determining the optimum sequence and number of amino acids for optimum binding to LPS can be determined with no more than routine experimentation. Segments of SEQ ID NO. 3 can be readily synthesized and binding experiments with, for example, immobilized P. aeruginosa lipopolysaccharide core can be carried out. The assay can be direct, as with radioactive labeled fragments of SEQ ID NO. 3 or can be indirect such as using competitive binding assays between SEQ ID NO. 3 and fragments of SEQ ID NO. 3. The invention thus contemplates the essential and optimal binding fragment of SEQ ID NO. 3.
The isolated lipopolysaccharide-binding fragments of a cystic fibrosis transmembrane conductance regulator can be labeled or coupled to a drug for targeting P. aeruginosa in vitro or in vivo. The anti-Pseudomonas drug can be any drug effective in either diagnosing a Pseudomal infection or in treating a Pseudomal infection. Such drugs include antimicrobials, antimicrobial potentiating agents, immune system recognition enhancers, diagnostic molecules and the like.
Molecules useful as antimicrobials can be delivered by the methods and compositions of the invention, such that the pathogenic infection is reduced or eliminated. Anti-Pseudomal agent drugs can be bactericidal or bacteriostatic by inhibiting replication of bacteria or inhibiting synthesis of bacterial components required for survival of the infecting organism. Anti-Pseudomonas antibiotic include the following: Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride; Carbenicillin Disodium; Carbenicillin Indanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium; Carumonam Sodium; Cefactor; Cefadroxil; Cefamandole; Cefamandole Nafate; Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride; Cefetecol; Cefixime; Cefinenoxime Hydrochloride; Cefmetazole; Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium; Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine; Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuoroxime Pivoxetil; Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; Cephalexin Hydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol; Chlortetracycline Bisulfate; Chlortetracycline Hydrochloride; Ciprofloxacin; Ciprofloxacin Hydrochloride; Colistin Sulfate; Coumermycin; Coumermycin Sodium; Doxycycline; Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; Droxacin Sodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride; Imipenem; Kanamycin Sulfite; Meclocycline; Minocycline; Minocycline Hydrochloride; Nafcillin Sodium; Norfloxacin; Ofloxacin;Oxytetracycline; Oxytetracycline Calcium; Piperacillin Sodium; Pirbenicillin Sodium; Tetracycline; Tetracycline Hydrochloride; Tetracycline Phosphate Complex; Ticarcillin Cresyl Sodium; Ticarcillin Disodium; Ticarcillin Monosodium; Tobramycin; and Tobramycin Sulfate.
The foregoing molecules may be coupled to CFTR or the fragment of CFTR that acts as the targeting moiety by the methods as described above in connection with the LPS-core targeting moiety.
The invention also involves isolated nucleic acids that encode the fragments of CFTR described above. Such nucleic acids are useful in producing the fragments described above. The nucleic acids can be part of an expression vector and can be included within a host cell. Virtually any cells, prokaryotic or eukaryotic, which can be transformed with CFTR DNA or RNA and which can be grown or maintained in culture may be used in the practice of the invention. Examples include bacterial cells such as E. coli and mammalian cells such as mouse, hamster, pig, goat, primate, etc. Specific examples include CHO cells and COS cells. Cell-free transcription systems also may be used in lieu of cells.
When used therapeutically, the compounds of the invention are administered in therapeutically effective amounts. In general, a therapeutically effective amount means that amount necessary to delay the onset of, inhibit the progression of, ameliorate the symptoms of or halt altogether the particular condition being treated. It is less than that amount that produces medically unacceptable side-effects. Generally, a therapeutically effective amount will vary with the subject""s age, condition, and sex, as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art. The dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days.
The therapeutics of the invention can be administered by any conventional route, including injection or by inhalation. The administrations may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. When the subject has cystic fibrosis, a preferred route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the therapeutic, such as the binding capacity of the polysaccharide (see, for example, Sciarra and Cutie, xe2x80x9cAerosols,xe2x80x9d in Remington""s Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those skill in the art can readily determine the various parameters and conditions for producing aerosols without resort to undue experimentation.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer""s dextrose, dextrose and sodium chloride, lactated Ringer""s or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer""s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
The invention also contemplates gene therapy. The procedure for performing ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents. In general, it involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject. The functional copy of the gene is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cell(s). Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654. In vivo gene therapy also is contemplated according to the invention. Gene therapy for cystic fibrosis is underway in clinical trials, and various vectors for expressing CFTR are known to those skilled in the art.
Certain of the various objects and advantages of the invention are illustrated in the following examples. Numerous equivalents and embodiments will be apparent to those of ordinary skill in the art and are intended to be embraced by the appended claims.